1. Field of the Invention
Dihydro and hexahydro isoalpha acids having a high ratio of trans to cis isomers, process for the production thereof, and products containing the same.
2. Prior Art
There are four types of isoalpha acids: the unreduced form, called isoalpha acids (isohumulone) (IA), and three types of reduced forms of IA. The latter are dihydro-isoalpha acids (DHIA), also known as xe2x80x9crhoxe2x80x9d, tetrahydro-isoalpha acids (THIA), and hexahydro-isoalpha acids (HHIA). Each is present as three major analogues differing in an acyl side chain (the co, n, and ad analogues) and as trans and cis and optical isomers. The proportions of analogues depends upon the variety of hops used to make the iso acids. Only IA, DHIA, and THIA have been and are available as aqueous forms. Their structures are shown in FIGS. 1 and 2.
IA and THIA do not form insoluble crystalline precipitates upon standing, due to their chemical composition, which includes a keto group on the lower acyl side chain. Commercially available all cis isomer DHIA and HHIA have this keto group reduced to an alcohol. They form precipitates over time, which are exceptionally hard to redissolve. Their solubility in water at pH 10 is about 1%, and much less at pH 7 to 8. The products described herein, containing large amounts of the trans isomers of DHIA and HHIA, are remarkably and unexpectedly soluble in water and overcome this limitation, being soluble in water at all concentrations below about 10% to 40%, depending upon the trans isomer content.
Today, the four types of iso acids used by the brewer are liquids, consisting of their potassium salts in water or propylene glycol. Solids in the form of magnesium chelates have been substantially replaced by the liquids in the last decade.
Because of differences in the concentrations at which the solutions of a particular type of iso acid are most stable against precipitation, the four acid types are sold in different concentrations in different solvent systems. IA is sold as a 30% solution of its potassium salt at a pH of about 10 in water. DHIA is sold as a 35% solution of its potassium salt in water at a pH of about 10.5 and above, from which large, insoluble crystals of DHIA will precipitate over time. THIA is used as a 5% or 10% solution of its potassium salt at a pH of about 9.5 to 10.5 in water; and HHIA is not sold as an aqueous solution per se because of its limited solubility. Because of the keto groups in their side chains, neither IA nor THIA form crystals from saturated solutions, but rather can form gums at the bottom of the container upon cooling and standing. In these commercial preparations, the hop acids, and particularly 30% IA and 35% DHIA, as potassium salts at pH 10 or above in water, act as co-solvents for themselves. The co-solvent effect is demonstrated by the known tendency to precipitate and separate at lower concentrations, as discussed below under the Westermann prior art. However, all forms of hop acids can be solubilized in propylene glycol, as described in Todd (U.S. Pat. No. 3,486,906), and are available in this form, which also adds the advantage of increasing their dispersibility in soft water at pH 10 and above. Propylene glycol and ethanol solutions are the only forms of HHIA available, and their utility is impaired by the requirement of a solvent. The high trans products overcome the need to use propylene glycol or ethanol as a solvent. It should be noted that soft water must be used as the diluting agent for all potassium salt solutions of the iso acids, since calcium and magnesium in the water will form chelates with hop acids and cause a haze and agglomerates and gummy precipitates. Below pH about 9 to 10 in deionized water, the dilute solutions of the prior art DHIA and HHIA do not form a clear solution upon mixing but rather form gummy precipitates upon standing. The high trans products do not.
One common method of adding the hop acids post-fermentation is to dilute them to a 1% or less concentration in soft water to which KOH has been added to bring the pH to 10 or above (Held, Master Brewers of the Americas Association Tech. Quarterly, 35, 132-138, No. 3 (1998). The high pH of the water is essential to prevent the formation of precipitates in the 1% dilute solution, and this has been ascribed to incomplete solubility of the hop acids in the dilute aqueous solution at lower pHs. These dilute alkaline solutions form hazes upon standing, and also form precipitates causing haze after injection into beer or xe2x80x9cstringersxe2x80x9d of precipitates on the inside of a pasteurized beer bottle. The viscosities of the concentrated solutions make it impractical to inject them directly into beer, and in addition they tend to xe2x80x9cshock outxe2x80x9d and form particulate matter due to the rapid reduction of pH as they are introduced, plugging the injection nozzle from time to time.
Solid magnesium chelates of IA are well described in Clarke, (U.S. Pat. No. 3,765,903 and 3,956,513). Others have added to his basic concept, but all IA chelates behave similarly. Chelates of DHIA and HHIA have never been commercialized. The water-insoluble microparticulate solid chelates are added to water, in which they disperse as a cloudy haze which in turn is added to the unfinished beer. Other chelate preparations are described in Humphrey (U.S. Pat. No. 3,875,316) and Mitchell (U.S. Pat. No. 1,161,787).
Aqueous suspensions of solid micro particles of DHIA and HHIA are described in Guzinski (PCT/US97/04070). These suspensions were made from commercial all cis products (p12, 1 23-24) made by the prior art procedures described herein. They are suspensions. They had the advantage over the prior art commercial solutions of DHIA in that they did not require heating to about 80-90xc2x0 C. to redissolve precipitates before use. Indeed, one of the major advantages was the ability to redissolve the micro-particles by heating to about 60xc2x0 C. The redissolved solution was in turn diluted to 1% in soft water at a pH of 10 prior to injection in the beer. Alternatively, the micro particles could be added directly to pH 10 soft water preheated to about 50xc2x0 C., wherein they would dissolve and form a clear 1% solution within five minutes. The 1% solution, as in the case of the other prior art commercial products, forms a haze upon standing (page 23, line 5), while the high trans product does not. Furthermore, his product, like other prior art products, is not soluble at a 1% concentration in neutral soft water, whereas the products described herein are completely soluble. And furthermore, his product still required heating, albeit less vigorous than 80-90xc2x0 C. The novel high trans product can preferably be used at ambient temperature, including brewhouse cellar temperatures of 10xc2x0 C. or less. The commercialization of his product was abandoned because of its limitations in practical brewing use, and particularly the need to heat it and the lack of clarity upon dilution.
DHIA is made from alpha acids by isomerization and reduction using sodium borohydride, first described by Koch (U.S. Pat. No. 3,044,879). A superior process based on Koch was described in Westermann (U.S. Pat. No. 3,798,332), which used an extract made by his earlier invention (U.S. Pat. No. 3,558,326). Goldstein (U.S. Pat. No. 4,324,810) describes a method of making DHIA without the use of organic solvents. Today, manufacturers optionally separate the alpha acids from the remainder of the extract prior to isomerization and reduction, as described in Goldstein U.S. Pat. No. 4,767,640. These investigators produced the essentially all cis forms of the acids.
Todd (U.S. Pat. No. 4,002,683) describes an improved method for separation of alpha acids and subsequent isomerization to IA, which is the preferred method of separating alpha acids from an extract. A less desirable procedure for the separation of alpha acids from an extract and conversion to IA is given in Klingel, (U.S. Pat. No. 3,364,265), who also describes solid salts of IA. Mitchell (U.S. Pat. No. 3,949,092) describes a superior process. The method of separating and purifying the alpha acids is not critical to the disclosed process. The purity of the reduced product is a critical element. As the Examples also show, the ratio of trans to cis isomers is very critical, and new to the art.
Procedures for making THIA from alpha acids are described in Stegink (U.S. Pat. No. 5,296,637), and Hay, (U.S. Pat. No. 5,013,571). THIA is made from beta acids after the procedure of Worden, (U.S. Pat. No. 3,923,897). HHIA is made by borohydride reduction after the method of Todd (U.S. Pat. No. 4,666,731, Example 10), who employs less than half the molar equivalents of Worden, (U.S. Pat. No. 3,552,975) to achieve reduction in a highly alkaline medium. Hay also describes the catalytic reduction of cis DHIA to make cis HHIA. HHIA, like DHIA, must be substantially free of impurities if it is to form the novel product of the present invention. None of these investigators have suggested this aspect of the present invention.
Guzinski (U.S. Pat. No. 5,200,227) describes mixtures of the prior art concentrated aqueous products which, due to co-solvent effects, do not readily crystallize. These had the advantage of physical stability over the single acid products, but imposed limitations on the ratios of different acids which the brewer could add to a beer. Occasionally, it was found that large crystals would form from these mixtures after prolonged storage, but not to the extent formed in the single-acid forms of commerce. Because of the limitations on the ratios of the different hop acids, they have limited utility. These products formed two phase, gummy solutions upon dilution in water, just as do prior art 35% DHIA solutions. The novel forms of DHIA and HHIA described herein overcome these limitations, since they are non-crystallizing and do not form gummy particulates.
Bavisotto (U.S. Pat. No. 3,615,660) describes the use of emulsifiers to stabilize DHIA extracts and make them suitable for adding to wort or beer. The instant products overcome the need for the use of emulsifying agents which end up in the beer, and the precipitation of the DHIA extract as the emulsion breaks upon addition to the beer.
Ting and Goldstein J. Am. Soc. Brew. Chem. 54, 103-109 (1996) describe the chemistry and purification of hop acids and their derivatives. Their investigation examined specific pure cis and trans isomers. They further described the physical properties of certain of these isomers. They did not evaluate the solubilities of their pure compounds , including their crystalline compounds and mixtures of them in water. They did not have, or suggest, the novel high trans isomer content aqueous solutions as described herein, containing all of the analogues of the parent hop.
While the primary function of hops is to provide bittering to beer, a secondary function is to provide aroma. The aroma is derived from the essential oil contained in the hop cone. Aroma control is compatible with this invention by addition of hop essential oil to the kettle (preferably in the saponified extract described in Guzinski, U.S. Pat. No. 5,750,179). This invention also allows the addition of hop essential oil to the DHIA and HHIA solutions, wherein it is sufficiently soluble to enable the brewer to add controlled amounts of essential oil to the finished brew.
Held, cited above, summarizes the status of prior art hopping methodology.
The objects of this disclosure are to provide DHIA and HHIA having a high trans to cis isomer ratio and, as a consequence, to provide:
1. A non-precipitating solution of DHIA and/or HHIA.
2. Non-precipitating mixtures of DHIA and/or HHIA solutions with added IA and THIA.
3. Hop acid solutions which do not form a haze or particulates upon direct injection into finished beer.
4. The analytical criteria which will provide quality assurance for the products, and which differentiates them from all prior art products.
5. The operational variables which may be adjusted by the manufacturer when making the novel products.
This specification discloses DHIA and HHIA having a high ratio of trans to cis isomers and which form clear, non-precipitating aqueous solutions of DHIA and HHIA, both of which are unknown to the prior art. This is due to the heretofore unknown effect of the trans isomers in increasing the solubility of the cis isomers. There is no explanation of this effect, which is contrary to the expectation that higher solute contents decrease solubility of all solutes. This effect is noticed in both neutral and slightly (up to pH 10-11) alkaline water. Because of the improved solubility in relatively low pH aqueous media and beer, the ease of use and utilization in the brewery is vastly improved as compared with the prior art cis products.
They do not form precipitates which must be heated to redissolve, or which must be filtered from the beer. They are soluble in soft water and their dilute solutions will not form hazes in the brewing cellar injection tank. Because the purity of the hop acid must be high to make them clear, they do not contribute an off-flavor xe2x80x9changxe2x80x9d to the beer, but rather possess only the desired fleeting bitterness without after-bitter, especially on the palate. They can be directly injected into finished beer without forming haze or visible particulates, contrary to prior art products.
The preparation of the product critically differs from the prior art in that the reduction is performed in an aqueous medium with sodium borohydride (potassium borohydride is less preferred) at a pH below about 12, and preferably in the range of about 10 to 11, and at temperatures, times and concentrations which do not convert trans isomers to cis isomers. Prior art products are made using a more highly alkaline aqueous medium (pH 13.5), since it is well known that borohydrides decompose readily if the water in which they are dissolved is not highly alkaline. The presently-disclosed and critical procedure allows some borohydride to decompose due to the lower pH, while the remainder acts as a reducing agent. Buffers may be used to achieve relatively stable pHs during the reduction.
The effect of the lower pH on the DHIA or HHIA is to allow trans isomers to form without being changed to the cis isomer. It increases the critical ratio of trans to cis isomers. Unless the trans isomer HPLC area count is at least 10% of the cis isomer area count, and preferably greater than about 20 to 30%, the product will not form a clear liquid aqueous solution at all concentrations from 1% to 20% and more. This is critical to the invention. Prior art products have a ratio of trans to cis isomers of less than about 3% to 5% and, in most, trans isomers are undetectable. None of them will form clear solutions at concentration ranges of 10-20% in water, even at elevated pHs. The novel solubility properties of high trans isomer ratio containing DHIA and HHIA are disclosed for the first time in this specification.
The preferred method also involves the reduction of IA rather than alpha acids. This increases the trans isomer ratio more than if a simultaneous isomerization/reduction is performed, as is the common prior art practice. The simultaneous isomerization-reduction does not produce an acceptable product.
The most elegant prior art investigations of DHIA have been done at the Miller Brewing Co. laboratories.
The initial disclosure of a process for making DHIA is Koch, cited above, filed in 1959. His examples use more than three to four times as much borohydride as the current art. Improved analytical techniques have enabled his Miller successors to refine his basic process. Koch""s DHIA products were dissolved in ethanol and added to boiling wort, so they obviously were not suitable for post-fermentation addition.
The Westermann series built on Koch, and developed practical processes for making DHIA using simultaneous isomerization/reduction. More importantly, in U.S. Pat. No. 3,965,188, they showed how to make DHIA solutions suitable for post-fermentation addition because of higher purity than achieved by Koch, wherein xe2x80x9cthe purity is so high (at least 90%) that the increase in turbidity is minimalxe2x80x9d. (Col. 2, line 10 ff). His procedures, because of the use of SWS (12% NaBH4 in 40% NaOH), does not make a high trans DHIA but rather an all cis one, which will form precipitates upon standing. This is why the high trans product cannot be made by Westermann""s U.S. Pat. No. 3,558,326. He claimed purities of between 97.4 and 99.2%. Example 13 shows that his product is 77 to 78% rather than 99.2% DHIA by the standards described in this specification. Nor is it haze free, as is the product described herein.
It must be recognized that his xe2x80x9cpuritiesxe2x80x9d were determined by the best method available at the time, which consisted of extracting the xe2x80x9cpurexe2x80x9d DHIA from its alkaline solution into a water immiscible solvent, removing the solvent, and assaying the solids in alkaline methanol. The standard procedure at that time was to determine the absorbance at 254 nm of an alkaline methanol solution of the solids, and calculate the DHIA content using some extinction coefficient (not mentioned in his specification). Regardless of the value of that coefficient, his solids would have contained some humulinic acids, as well as other materials having absorbance at 254, and they would have been considered DHIA by his assay. Furthermore, as shown in the comparative Example 11, his product formed cloudy solutions at pH 10 in water, and curds and precipitates at pH 7. It did not contain trans isomers.
Goldstein, following Westermann at the Miller Brewing laboratories, also performs a simultaneous isomerization/reduction in U.S. Pat. No. 4,324,810. He also uses SWS, a commercial 12% sodium borohydride solution in 40% NaOH, and therefore his reduction is carried out under highly alkaline conditions which cause only cis isomers to form. His Examples 4 plus 5 show an overall yield of 82.7% of available DHIA with a purity of 96%. Not only was this an improvement on the yields of Westermann, but he achieved his paramount objective of performing the reduction without the use of solvents other than water. Again, the precise method by which he obtained his purity estimate of 96% is not given. As comparative Example 14 shows, his product was 74% DHIA vs his claimed 96%, by the state of the art techniques used in describing the purity of DHIA in this specification. His product did not contain trans isomers, nor did it form clear 1% solutions.
Goldstein in U.S. Pat. No. 4,767,640 separates the alpha acids from the extract, at a marginally higher pH than the critical pH of Todd, prior to isomerization/reduction without the use of solvents. He obtains an improved product, devoid of non-isohumulone light unstable products (NILUPS) found in the prior art products. (While Westermann claimed complete light stability, it is clear that the detection of instability had progressed by the time of Goldstein""s invention, and he was able to improve the light stability of Westermann""s products.) His product is claimed suitable for post-fermentation addition to beer, but not specifically for pre- or post- final filtration. This may be because his product forms amorphous agglomerates and crystals on standing. It does not contain trans isomers. This is because his isomerization/reduction, as in Westermann, is conducted in a highly alkaline medium to start with. Comparative Examples 15 and 16 describe his products. Injection of his products into finished beer cause insoluble precipitates to form. These are visible to the naked eye even after pasteurization.
While Goldstein prefers to avoid the use of solvents in his process, innocuous solvents such as hydrocarbons C-10 and below are useful in assisting the separations and purifications of the high trans products. They are not essential but rather optional and will assist in the removal of the unwanted impurities, some of which are visible as post hop acid peaks in the HPLC. Others, such as xe2x80x9cwaxesxe2x80x9d, may be undetectable in the HPLC assay. These must be substantially absent for the claimed DHIA and HHIA to remain clear in aqueous solutions when added to soft water.
Chicoye et al, in U.S. Pat. No. 4,759,941, describe a method for making DHIA by treating hop pellets with borohydride. From his reaction mixture, he is able to separate an aqueous fraction which he adds post kettle. He makes no claim that it can be added to finished beer, and therefore does not suggest the products described herein. Surprisingly, when pure alpha acids were reduced following his procedure, the reduction was incomplete and substantial impurities were formed. Perhaps his cellulosic materials act as a catalyst for the reaction to produce high by-product levels in his procedure. Trans isomers were not detected in his reactive product from alpha acids.
Guzinski""s all cis microcrystalline product, which requires heating to redissolve, either by itself or in alkaline water, is clearly not a relevant prior art disclosure. Likewise, his slowly precipitating mixtures of hop acids, which utilize their cosolvent effect, but are all cis isomers, do not suggest that the presence of trans isomers inhibits and prevents the crystallization of cis isomers. Nor do the solids of Clarke. HHIA is not available as an aqueous product, since the all cis form, made by the Todd procedure (U.S. Pat. No. 4,666,731), is very insoluble.
Table 7-I in Example 7 summarizes the critical differences between products from the comparative Examples and the herein claimed process, as well as the effect a high trans isomer content has on solubility. Table 9-I in Example 9 shows the differences in performance of the products in beer.
What we believe to be our invention, then, inter alia, comprises the following, singly or in combination:
A mixture of hexahydro-isoalpha acids (HHIA) or dihydroisoalpha acids (DHIA) having a ratio of trans to cis isomers greater than 10%.
And a mixture of hexahydro-isoalpha acids (HHIA) having a ratio of trans to cis isomers greater than 10%;
such a mixture comprising hexahydro-isocoalpha acids, hexahydro-iso-n-alpha acids, and hexahydro-isoadalpha acids;
such a mixture wherein the ratio is greater than 20%;
such a mixture wherein the ratio is greater than 40%; and
such a mixture wherein the ratio is greater than 70%.
Also, such a mixture in the form of an aqueous solution of potassium salts of the HHIA, which solution forms a single phase liquid at a 20% concentration by weight of the potassium salts at a pH less than 9.5;
such a mixture wherein the solution forms a single phase liquid at a 10% concentration by weight of the potassium salts of the HHIA at a pH less than 8.5;
such a mixture in the form of an aqueous solution of the potassium salts of the HHIA at a pH of 7 to 10.5 which is a single-phase solution when at a concentration of 5% by weight;
such a mixture in the form of an aqueous solution of the potassium salts of the HHIA at a pH of 7 to 9.5 which is a single-phase solution when at a concentration of 10% by weight; and
such a solution which, when diluted to a 1% concentration by weight in distilled water, forms a clear solution which does not form a haze upon standing for six hours.
Also, such a mixture which contains less than 5% by weight of substances which elute after the HHIA as detectable as area percent by HPLC procedure;
such a mixture which contains less than 3% by weight of substances which elute after the HHIA as detectable as area percent by HPLC procedure;
such a mixture which contains less than 1% by weight of substances which elute after the HHIA as detectable as area percent by HPLC procedure;
such a mixture which contains less than 3% by weight of the HHIA of substances which can be removed from an aqueous solution of the HHIA by extraction into a hydrocarbon solvent of 6 to 10 carbon atoms;
such a mixture which contains less than 2% by weight of the HHIA of substances which can be removed from an aqueous solution of the HHIA by extraction into a hydrocarbon solvent of 6 to 10 carbon atoms;
such a mixture which contains less than 1% by weight of the HHIA of substances which can be removed from an aqueous solution of the HHIA by extraction into a hydrocarbon solvent of 6 to 10 carbon atoms; wherein the pH of the aqueous solution is below 10.5; wherein the pH of the aqueous solution is below 9.5; and wherein the pH of the aqueous solution is below about 8.5.
Such a solution admixed with a solution of DHIA or with isoalpha acids (IA) or tetrahydroisoalpha acids (THIA);
such a solution containing glycerine, propylene glycol, alcohol, or hop essential oil; and
such a mixture in the form of solid potassium salts of the HHIA comprising between about 10% and 70% trans isomers.
And a mixture of dihydro-isoalpha acids (DHIA) having a ratio of trans to cis isomers greater than 10%;
such a mixture comprising dihydro-isocoalpha acids, dihydro-iso-n-alpha acids, and dihydro-isoadalpha acids;
such a mixture wherein the ratio is greater than 20%;
such a mixture wherein the ratio is greater than 30%.
Also, such a mixture in the form of an aqueous solution of potassium salts of the DHIA, which solution forms a single phase liquid at a 20% concentration by weight of the potassium salts at a pH less than 9.5;
such a mixture wherein the solution forms a single phase liquid at a 10% concentration by weight of the potassium salts of the DHIA at a pH less than 8.5;
such a mixture in the form of an aqueous solution of the potassium salts of the DHIA at a pH of 7 to 10.5 which is a single-phase solution when at a concentration of 5% by weight;
such a mixture in the form of an aqueous solution of the potassium salts of the DHIA at a pH of 7 to 9.5 which is a single-phase solution when at a concentration of 10% by weight; and
such a solution which, when diluted to a 1% concentration by weight in distilled water, forms a clear solution which does not form a haze upon standing for six hours;
Also, such a mixture which contains less than 5% by weight of substances which elute after the DHIA as detectable as area percent by HPLC procedure;
such a mixture which contains less than 3% by weight of substances which elute after the DHIA as detectable as area percent by HPLC procedure;
such a mixture which contains less than 1% by weight of substances which elute after the DHIA as detectable as area percent by HPLC procedure;
such a mixture which contains less than 3% by weight of the DHIA of substances which can be removed from an aqueous solution of the DHIA by extraction into a hydrocarbon solvent of 6 to 10 carbon atoms;
such a mixture which contains less than 2% by weight of the DHIA of substances which can be removed from an aqueous solution of the DHIA by extraction into a hydrocarbon solvent of 6 to 10 carbon atoms;
such a mixture which contains less than 1% by weight of the DHIA of substances which can be removed from an aqueous solution of the DHIA by extraction into a hydrocarbon solvent of 6 to 10 carbon atoms;
wherein the pH of the aqueous solution is below 10.5;
wherein the pH of the aqueous solution is below 9.5;
wherein the pH of the aqueous solution is below about 8.5.
Such a solution containing glycerine, propylene glycol, alcohol, or hop essential oil;
such a mixture in the form of solid potassium salts of the DHIA comprising between about 10% and 70% trans isomers.
Moreover, such a mixture of DHIA or HHIA which is in the form of a single-phase aqueous solution of its potassium salts at a pH above about 7.5 when at a concentration of 20% by weight.
Furthermore, the process of reducing (a) isoalpha acids (IA) to produce dihydroisoalpha acids (DHIA) or (b) tetrahydroisoalpha acids (THIA) to produce hexahydroisoalpha acids (HHIA), the DHIA or the HHIA product having a trans to cis isomer ratio greater than 10%, the reduction being carried out in an aqueous medium at a pH of about 8.5 to about 12.4 using a borohydride;
such a process wherein IA are reduced to DHIA having a trans to cis isomer ratio greater than 10% using less than about 0.81 molar equivalents of a borohydride and a pH up to about 11.8;
such a process wherein THIA are reduced to HHIA having a trans to cis isomer ratio greater than 10% using less than about 0.81 molar equivalents of a borohydride;
such a process in which the temperature at which the reduction is carried out is up to about 75xc2x0 C. and in which the reaction is terminated before the trans to cis isomer ratio of the product DHIA or HHIA becomes less than 10%;
such a process wherein the reduction is carried out with up to about 0.65 molar equivalents of borohydride;
such a process wherein the reduction is carried out with up to about 0.55 molar equivalents of borohydride;
such a process in which a lower alkanol is also present;
such a process wherein the pH of the aqueous medium is buffered at about 12.4 or below;
such a process wherein the buffering agent is selected from potassium and sodium salts of phosphates, citrates, and borates;
such a process in which a non-reactive water-immiscible solvent is also present;
such a process in which the water-immiscible solvent is a hydrocarbon containing 10 or less carbon atoms;
such a process in which hydrocarbon-soluble haze-forming substances are removed from the DHIA or HHIA product by admixing a hydrocarbon with the aqueous DHIA or HHIA phase and removing the hydrocarbon phase, wherein the aqueous DHIA or HHIA phase is 15% or less DHIA or HHIA, and wherein the pH is up to about 10.5, to give a DHIA or HHIA product wherein the remaining hydrocarbon-soluble substances are less than 3% by weight of the DHIA or HHIA product;
such a process in which the pH is about 7.5-9.5;
such a process wherein the hydrocarbon has 6 to 10 carbon atoms;
such a process in which the final aqueous DHIA or HHIA phase is concentrated at a pH below about 10.5 and greater than 6 by evaporation of water, to give a concentrated aqueous phase containing between about 5% and about 40% DHIA or HHIA;
such a process wherein the pH is between 6.5 and 8.5 and the DHIA or HHIA concentration is less than about 25%;
such a process wherein the borohydride is selected from the group consisting of sodium borohydride and potassium borohydride; and
such a process wherein the DHIA having a trans to cis isomer ratio greater than 10% is subsequently converted to HHIA having a trans to cis isomer ratio greater than 10% by catalytic hydrogenation.
Definitions Used in this Specification
As is known to the art, trans and cis isomers of the hop analogues exist. Critical to this invention is the heretofore unknown effect of a high trans:cis isomer ratio on the solubility of the DHIA and HHIA. In this specification, this ratio is expressed as the % of the HPLC area counts of the trans divided by the area counts of the cis isomers. In prior art products it is well below 5% and usually almost zero.
One measurement of % impurities eluting after the hop acids in the HPLC procedure is described below. It expresses the amount of haze forming substances, which are detectable by uv light, present relative to the amount of DHIA or HHIA. A second measurement relies on the extraction of non-absorbing haze forming substances with a water insoluble solvent, as described in Example 11 below.
Procedures Used in Analysis are as Follows
Ultra-violet spectra (UV). For a whole extract, the American Society of Brewing Chemists spectro procedure xe2x80x9cHops-6xe2x80x9d was used. This entails diluting the test sample in alkaline methanol and running a scan, and using a formula to calculate % alpha acids. This procedure is included in the prior art references.
Absorption at 254 nm is a maximum for iso acids, and the strength of the sample is calculated on this basis using the extinction coefficient (E1%/1 cm). The sample is dissolved in alkaline methanol, the absorbance at 254 nm determined, and the concentration calculated from the extinction coefficient. This procedure registers all absorbance at 254 nm as the iso acid, and if absorbing impurities, such as humulinic acid, are present, it therefore overstates the true iso acid content. Only by HPLC can the true value of hop acid concentration be determined.
The extinction coefficient will vary for the various hop acids due to differences in molecular weights, analogue composition, and the standards historically used to determine them. For the purposes of this specification, the following numbers are used:
Experimental Method for HPLC Measurements
Hop extracts are diluted to a concentration of about 200-500 ppm total hop acids in methanol. Separations are performed on a Waters 2690 Separations Module with a 996 Photodiode Array. The HPLC column contains octyl reverse phase packing (Zorbax Eclipse XDB-C8, 25xc3x970.46 cm, 5-micron) and was kept at 25xc2x0 C. The aqueous buffer is 18:82 (v/v) acetonitrile:1% aqueous citric acid buffer (pH 7.0). The citric acid buffer is prepared separately, adjusted to pH 7 with 45% KOH, and filtered before combination with the acetonitrile. The mobile phase program is given in Table D-1. Injection volume is 5 xcexcL.
The detector is set to measure the entire UV absorbance spectrum between 230-400 nm with a resolution of 1.2 nm, filter response of 1, and sampling rate of 1 point/sec. HPLC plots are reported in xe2x80x9cmaxplotxe2x80x9d mode, which reports the maximum absorbance value between 230-400 nm at each point in the chromatogram. Data is analyzed by Millennium(copyright) 32 software (version 3.05.01, Waters and Associates). Maxplot chromatogram peaks are quantified with integration settings of threshold=15 xcexcV/s, filter response=1, and minimum height and area=0.
The % impurities eluting after the hop acid is determined using the % area count at peak maximum. This is because many of the impurities do not have significant absorbance at 254, but peak in the range of 270 nm and above. An extinction coefficient is not needed for this calculation, as it only measures the total area under the peaks at the absorption maximum. The subject hop acids are identified in the traces, as well as the peaks eluting after them, and the instrument calculates the area counts. The relative area counts are independent of concentration of the solution injected into the HPLC.
The cis and trans isomer peaks are defined in the HPLC traces of FIGS. 3 to 6 for DHIA, and FIGS. 7 to 10 for HHIA. Since the prior art has not investigated the relationship of these peaks, the authors have designated these peaks as trans or cis, as defined in Example 10. The definitions provided by the Figures show the critical differences between the prior art and the novel products described in this specification.
Haze is measured by the American Society of Brewing Chemists procedure Beer 26.
Equivalents of a substance are molar.
Yields are based on an average molecular weight of the mixture of analogues.
Infra-red (IR) spectra are useful for demonstrating the different chemical composition of the pure hop acid and the haze forming substances which do not absorb uv light. For the purposes of this specification, they are defined as xe2x80x9cwaxes.xe2x80x9d These are isolated and the spectra described in Example 11.
The Novel Process and Product
Examples 1 thru 4 show variations on the preferred process for making a high trans , highly soluble DHIA and HHIA which, in turn, do not form hazes upon dilution to 1% in distilled water..
It will be noted that none of these products form insoluble precipitates on standing, and that they may be added directly to soft water to whatever concentration the brewer desires. It will also be noted that they do not form precipitates visible to the unaided eye or measurable haze upon direct injection into bottled beer. None of the prior art products have these qualities. Goldstein""s NILUPs-free type DHIA all cis products and the all cis HHIA products presently available do not form clear solutions.
The purity of the hop acids must be exceptionally high if solutions of the high trans product are to remain clear. Substances eluting after the hop acid in the HPLC procedure must be less than 6%, preferably less than 4%, and most preferably less than 1% to 2%. Likewise, xe2x80x9cwaxesxe2x80x9d which do not absorb uv light and which are hexane soluble, must be less than 3%, preferably less than 2%, and most preferably less than 1%.
It is well known to the art that different hop varieties produce different ratios of the three major alpha acid analogues. The lower molecular weight analogues have more solubility than the higher molecular weight ones. As a consequence, the upper concentration limit of the high trans products will vary with hop variety. The concentrations shown in the Examples are considered to be economical to the brewer and suitable for any variety with which the authors are familiar.
The authors can offer no theory as to why a trans to cis isomer ratio of above about 10%, especially above about 20% to 30%, results in the greatly increased solubility of the cis isomers, which, as mentioned in Example 6, are shown to be about 1.5% maximum for equally pure cis DHIA and 0.75% for cis HHIA. In some unknown manner, the trans isomers increase the solubility of the cis isomers from about 1% in water to 10% and more. For example, a 20% solution of HHIA containing 4% trans isomers and 16% cis isomers does not form crystalline precipitates. A 16% solution of cis isomers does. As mentioned above, the solubility of the cis isomers alone is about 1%. The effect of the trans isomers upon the solubility of the cis isomers"" solubility is contrary to expectation, since the higher the solute content, the lower should be the solubility of related compounds. This effect cannot be a simple result of the analogue mixture, since the analogues are the same for the cis and trans forms. However, it is also preferred that the claimed products contain the approximate mixture of analogues found in the parent hop. Neither of these critical elements-the high trans isomer ratio combined with all of the parent hop analoguesxe2x80x94have grounding in the prior art.
Edible anti-freeze substances, such as ethanol, propylene glycol, and glycerine may be added to the inventive products if they are to be exposed to below freezing temperatures.
In the process, buffering agents other than potassium phosphate may be used. These include sodium and other phosphates, as well as borates and citrates. Details concerning the required process parameters are discussed in the Examples.
When the claimed products are dried, they form amorphous solids which can readily be rehydrated to form aqueous solutions with the properties of the original aqueous solutions. Dehydration can be performed by techniques known to the art, such as spray drying or by evaporation of water under vacuum or even at atmospheric pressure.
The claimed product is differentiated from prior art products by its high trans isomer content, the trans isomers being at least about 10% of the cis isomers (a trans to cis ratio of 10%), and preferably 20%, and most preferably above 30%. It is further differentiated by the absence of substances which elute after the DHIA or HHIA by HPLC analysis, such substances consisting of artifacts and by-products of the reduction reaction. These substances interfere with the clarity of the aqueous solutions of the products. The products are further differentiated from the prior art in that substances which are soluble in hydrocarbon solvents and not detected by the HPLC procedure are essentially absent.
Furthermore, the products form stable single phase aqueous solutions at pHs substantially below the 10.5 to 11 minimums of the prior art, for example between about 7 and 9.5, and are not dependent upon a 35% hop acid concentration, as shown by Westermann, to make a pourable liquid product. The solutions are stable at concentrations in the range of about 5% to 40%. While the instant products are preferably maintained at a pH below about 9.5, they are also stable at the pHs of the prior art.
In addition, the products form clear, non-hazing solutions in distilled water at concentrations of 1% to about 5% and more. Prior art products require raising the pH of the water to above 10 to effect dispersion of a 1% solution, and even then haze forms upon standing. They form gums and precipitates when added to distilled water. This simple test is one means of determining if the product meets the analytical requirements described above and in the claims.
The procedures by which these products are made combine elements of the prior art in a new way, so as to achieve the high trans ratio product. Unlike the Koch and Worden prior art, which uses about two or more molar equivalents of borohydride to achieve reduction and light stability, the herein disclosed novel procedure requires less than about 0.81 molar equivalents. Unlike the prior art conventional pHs of above 13 of Westermann, Todd, Goldstein, and others, who also use less than 0.81 equivalents, the pH during the reduction must be below about 12.4, preferably below about 12.2, and optimally below a pH of 11.2 or even 10.6 for THIA. For IA, the upper pH should be below about 11.8, and preferably below 11. While these pHs, which are well below the prior art, result in some borohydride decomposition which the prior art pHs above about 13 to 13.5 deliberately avoided, the low pH is critical to the trans isomer formation. The high pH of the prior art resulted in essentially all cis products, which are inherently of very low solubility. As little as 0.4 molar equivalents of borohydride may be used, but the range of about 0.55 to 0.65 is preferred. When the large excesses of borohydride, such as shown in Worden, are used, over-reduced and other by-products are formed and the actual yield of reduced DHIA or HHIA is so small as to make analysis problematic and the elimination of haze forming substances very difficult if not impossible.
Reaction temperatures below about 85xc2x0 C. are feasible, the reaction taking longer at lower temperatures. The preferred range is about 40xc2x0 to 75xc2x0 C.
The reduction should be terminated before a significant amount of trans isomer is converted to the cis form. This occurs more rapidly at high pHs and temperatures. The analytical procedures described herein provide a guide to termination times.
Combining purification steps with the novel reduction conditions discloses how heretofore unidentified haze and precipitate forming substances can be removed. These purification steps address substances eluting after the DHIA or HHIA by the HPLC procedure (see Definitions and Example 7 of this specification). These post-eluting substances must have a total area count at peak maximums, according to the HPLC procedure, of less than 5%, preferably less than 3%, and most preferably less than 1% of the area counts of the hop acids.
In addition, there are also non-uv absorbing substances, undetectable by the HPLC procedure, which must be critically less than about 3%, preferably less than 2%, and most preferably less than 1% of the weight of the hop acids.
Removal of these unwanted and unidentified substances, called xe2x80x9cwaxesxe2x80x9d in this specification, and not absorbing uv light, is preferably achieved by separating them from aqueous solutions at a pH below 10.5, and preferably below about 8.5 to 9.5, and even as low as 7.5. The concentration of the hop acids in the aqueous phase during xe2x80x9cwaxxe2x80x9d removal is less than about 20%, and preferably less than 15%. Because of the insolubility of the all cis prior art forms at these pHs, separations were done at elevated temperatures (Goldstein) or less than about half of the DHIA was captured into the xe2x80x9ccleanxe2x80x9d phase. As shown by the comparative Westermann and Goldstein examples, yields were poor and sufficient impurities were present to cause haze and precipitation when diluted in distilled water. It is speculated that the presence of the trans forms assists in the separations, and therefore is critical to the xe2x80x9cclean-upxe2x80x9d procedure. The herein disclosed art gives yields in excess of 75% and up to 85% to 90%.
Separation of unwanted substances is preferably effected using a hydrocarbon solvent, especially of C-6 to C-10, but other water immiscible solvents such as ether or methylene chloride may be used. Less preferably, they may be separated by allowing agglomerates of the substances to form, optionally in the presence of solid adsorbents such as diatomaceous earth, and filtering the solids from the liquid phase. As with the water immiscible solvents, the solid separations are conducted at a pH below about 9.5.